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Toward an in-situ glucose biosensor for animal cell culture monitoring and control

University of British Columbia

This thesis describes the development and characterization of an in-situ sterilizable, regenerable biosensor for monitoring and control of glucose concentration in bioreactorscale animal cell culture. Long-term stability and in-situ steam sterilization capability are obstacles preventing development of industrially acceptable biosensors. These difficulties are magnified in animal cell culture applications given their longer duration and high susceptibility to microbial contamination. A novel enzyme immobilization technology is presented which helps address these difficulties. The developmental starting point utilized an existing prototype originally designed for microbial culture applications. The prototype contains a cellulose matrix sandwiched between a platinum electrode and a dialysis membrane. Glucose oxidase enzyme, chemically conjugated with cellulose binding domain protein, is easily immobilized to and removed from the cellulose. An improved conjugation protocol yielding conjugate with higher specific activity is described. Conjugate was characterized for binding characteristics to cellulose and lower detection limit during prototype use. Binding experiments were the first for characterizing a heterogeneous chemical fusion of these proteins. Re-design of the prototype membrane assembly and incorporation of continuous perfusion are described and were necessary modifications to produce a functional prototype for animal cell culture. Longer term sensor signal stability was characterized and enhanced. A first-order equation adequately described signal decay behavior during the first long-term experiment. The combination of hardware modifications resulted in ca. 2.5-fold increase in signal stability during controlled environment conditions. A subsequent cell culture experiment revealed enhanced signal stability; although, undefined factors resulted in inconsistent decay patterns. A Chinese hamster ovary cell line was transfected with pNUT plasmid expressing H6E2FX protein to develop a model cell line for culture and glucose studies utilizing the prototype. Positive subclones, after adaptation to serum-free medium over 7 weeks, displayed undetectable levels of H6E2FX. Several possible explanations could account for unstable expression but no specific one was determined. Described hardware modifications yielded a prototype functional for longer periods of time and with enhanced signal stability. Stability experiments are the first for characterizing longer term prototype signal behavior. Results in this thesis contribute to existing data demonstrating the feasibility and potential of this biosensor technology for implementation in industrial bioprocesses.

Thesis/Dissertation

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2009-05-23

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http://hdl.handle.net/2429/8080

Microbiology and Immunology

University of British Columbia

UBCV

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